INTRODUCTION
During development, fungi are often exposed to unfavorable growth conditions such as hypoxia or even anoxia. These environmental changes are bound to produce effects on cell energy metabolism, but such effects have been insufficiently studied.
Phycomyces blakesleeanus is a strictly aerobic fungus (Ceredá-Olmedo and Lipson, 1987) and therefore can be a good model system for studying the effects of anoxia. Among methods that can be used, 31P
NMR spectroscopy is uniquely suited for such stud-ies, since it permits nondestructive in vivo identifi-cation of phosphate compounds involved in energy metabolism.
When energy metabolism is considered, the ATP molecule is usually the first thing that comes to mind. For Saccharomyces cerevisiae, it was shown that ATP resonances in 31P NMR spectra slightly
decrease when yeast cells are transferred from aero-bic to anaeroaero-bic conditions (den Hollander et al., 1981; Campbell-Burk et al., 1987). Intensity of the
31P NMR signal of UDPG, which is a precursor for
the synthesis of storage carbohydrates and cell wall material and thus an indicator of cell growth
poten-tial, shows no significant changes in Candida tropi-calis, regardless of oxygenation (Lohmeier-Vogel et al., 1995).
In contrast to the signals of ATP and UDPG, major changes of 31P NMR spectra during anoxia are
observed in the signals of inorganic phosphate (Pi) and polyphosphates (PPn) (Pilatus and Techel, 1991; Beauvoit et al., 1991). It is the general opinion that PPn play an important role in fungal energy metabo-lism, since they regulate level of ATP and function as either a high-energy reserve or a phosphate reserve via hydrolysis (Harold, 1966). However, the effects of anoxia on intensity of PPn signals are diverse.For
S. cerevisiae, one group of results indicate that if the yeast cells are taken during the logarithmic growth phase, 31P NMR spectra recorded in oxygenated and
anoxic conditions show no significant differences (den Holander et al., 1981). However, if the yeast cells are taken during the stationary phase (when all glucose from the medium is used up), intensity of the PPn signal decreases in anoxic conditions, while that of the intracellular inorganic phosphate (Pi) signal increases (den Holander et al., 1981; Campbell-Burk et al., 1987). A second group of results indicate that
EffEcts of anoxia on 31P nMR sPEctRa
of PHYCOMYCES BLAKESLEEANUS duRing dEvEloPMEnt
MARINA STANIć1, M. ŽIVIć2, and JOANA ZAkRZeWSkA2
1Faculty of Biology, University of Belgrade, 11000 Belgrade, Serbia 2Institute of General and Physical Chemistry, 11000 Belgrade, Serbia
Abstract — The method of 31P NMR spectroscopy was used to investigate the effects of anoxia on Phycomyces blakesleea-nus mycelium during development. The greatest changes were recorded in the PPc, NADH, and α-ATP signals. Decrease of PPc signal intensity is due to chain length reduction and reduction in number of PPn molecules. Smaller decrease of β-ATP compared to α-ATP signal intensity can be attributed to maintenance of ATP concentration at the expense of PPn hydrolysis. Sensitivity to anoxia varies with the growth stage. It is greatest in 32-h and 44-h mycelium, in which PPn is used as an additional energy source, while the smallest effect was noted for 36-h fungi.
Key words: 31P NMR, anoxia, Phycomyces blakesleeanus, development, polyphosphates
UDC 582.281.24:581.12:544.176
Certain conclusions can be drawn from these results, in spite of their inconsistency. Transfer of yeasts to anaerobic conditions leads to synchronized changes in PPn and Pi concentrations, and these changes depend on the development phase. The absence of a similar response in N. crassa may be a result of species specificity of respiratory chain structure, since a possible link between PPn con-centration and mitochondrial energy metabolism has been established (Beauvoit et al., 1989), together with considerable complexity and diversity of the “respiratory network” in fungi (Milani et al., 2001).
In our previous paper (Živić et al., 2007), we showed that changes of the PPn to Pi signal intensity ratio are linked to characteristic stages of sporan-giophore development. The obtained results sug-gested a role for polyphosphates as an energy and/or phosphate reserve during P. blakesleeanus develop-ment. In view of the contradictory results obtained on the effects of anoxia on 31P NMR spectra in S. cerevisiae and N. crassa and their dependency on development, research on the effects of anoxia on P. blakesleeanus during different growth phases could provide an additional contribution to understanding fungal energy metabolism.
MATeRIALS AND MeTHODS
Strains and growth conditions of mycelia
The wild type strain NRRL 1555(-) (Burgeff) of the fungus Phycomyces blakesleeanus was used in this study. One milliliter of spore suspension containing around 106 spores was seeded in standard minimal
medium (Sutter, 1975): 36.7 mM kH2PO4, 2 mM MgSO4 x 7H2O, 0.376 mM CaCl2, 3 μM thiamine x HCl, 1 μM citric acid x H2O, 3.7 μM Fe(NO3)3
continuous overhead illumination with fluorescent light of 10 W/m2 at 22oC and ca. 95% relative
humidity.
NMR measurements
For NMR measurements, mycelia in specified growth phases (up to 10 Petri dishes were needed depending on the growth phase) were collected by vacuum filtration, washed with modified minimal medium (0.2 mM kH2PO4 without microelements, pH=4.65). An amount of 0.6-0.8 g (fresh weight) of mycelia was suspended in 2.5 ml of aerated modi-fied minimal medium and packed in a 10-mm NMR tube. The mycelia were harvested every 4 hours from 16 h of growth onward in order to characterize the main growth stages. After recording of four control spectra, the samples were bubbled with N2 for 5 min in order to replace the oxygenized atmosphere with an inert one. Four more spectra were taken in the oxygen-free atmosphere. The first (k1) and fourth (k4) control spectra, as well as the first (N1) and fourth (N4) spectra taken in anaerobic conditions, were used for analysis.
The 31P NMR measurements were performed
using a Bruker MSL 400 spectrometer operating at 161.978 MHz for 31P. Spectra were accumulated in
4k data points with 7-μs pulse duration and 300-ms recycle time using a spectral width of 11363 Hz. Line broadening of 25 Hz was applied before Fourier transformation. Methylene diphosphonic acid at 17.05 ppm relative to 85% H3PO4 was used as an external standard.
ReSULTS
The effects of anoxia on the 31P NMR spectra of
are shown in Fig. 1. The assignment of signals was made as in a previous paper of ours (Živić et al., 2007). It can be seen that for 36-h spectra, notewor-thy changes occur only in Pi signal intensity, which increases in anaerobic conditions by 31%, and in UDPG signal intensity, which decreases by 11%. Contrary to this, anoxia causes a large decrease in intensities of all signals in 44-h spectra except the Pi signal, whose intensity does not change. The largest decrease of intensity is observed for the PPc signal (58%).
Figure 2 shows changes in 31P NMR signal
inten-sity ratios in control conditions (k4/k1), anaero-bic and control conditions (N1/k4), and prolonged anaerobic conditions (N4/k4). Average signal inten-sity ratios for selected growth phases (Fig. 2A) and average signal intensity ratios of selected signals for all growth phases (Fig. 2B) are shown. In control conditions (k4/k1), only slight changes of signal intensity ratios (less than 10%) are observed, except for a statistically significant increase in Pi signal intensity of 17±5% and decrease in UDPG signal intensity of 21±2% (Fig. 2B).
Transfer to anaerobic conditions (N1/k4) causes decrease in intensities of all signals in the spectra except for Pi, whose intensity increases (10±7%, Fig. 2B). Decrease of intensities is statistically significant (p<0.05) for all signals except β-ATP and UDPG, the greatest declines occurring in the PPc, α-ATP, and NAD(H) signals. Also, the decrease of α-ATP signal intensity (p=0,048) is greater than that of β-ATP signal intensity. Average signal intensity ratios for selected growth phases (Fig. 2A) are lower than in the control, except for 16-h specimens, which show a statistically significant intensity increase (13±4%), and 36- and 28-h specimens, which show no sig-nificant changes after N2 treatment. The greatest decrease was obtained for 32- and 44-h fungi.
Prolongation of anoxia (N4/k4) results in further decrease of all signal intensity ratios except that of Pi, which remains constant. However, decrease in the signal intensity ratio is not statistically sig-nificant for the PPc and NAD(H) signals. The effects of prolonged anoxia on 31P NMR average signal
intensity ratios for selected growth phases are most distinct at 16 h (p=0.005), 40 h (p<0.001) and 48 h
Fig. 2. Changes in the 31P NMR signal intensity ratio of Phycomyces blakesleeanus in control conditions (K4/K1, ○, black line), an-aerobic and control conditions (N1/K4,, dashed line), and anaerobic conditions (N4/K4,, dotted line). (A) mean values of all signal intensity ratios for selected growth phase; (B) mean values of intensity ratios of selected signal for all growth phases.
between these two signals were further analyzed in different growth phases during anoxia (Fig. 3). The results presented in Fig. 3 show that anoxia induces a decrease of the PPc/Pi ratio, two groups dependent on the growth stage being discernible, the first (16-28 h) less sensitive, the second (32-48 h) more sensi-tive to anoxia. In the less sensisensi-tive group, the PPc/Pi ratio varies from 21% (16 h) to 35% (28 h), while in the second group it ranges from 48% (32 h) to 57% (40 h), the only significant exception being recorded for 36-h fungi (31%).
DISCUSSION
It was confirmed earlier that PPn play an important role in response to stress conditions like osmotic shock (Yang et al., 1993), changes in pH (Greenfield et al., 1987), and phosphate and glucose starvation (Bourne, 1991; kulaev and kulakovskaya, 2000). Having this in mind, it can be expected that PPn will be involved in the response to a strong stress like transition to anaerobic conditions. However, research conducted to date did not firmly confirm these expectations. In S. cerevisiae, it was established that anoxia induces decrease of PPc signal intensity and increase of Pi signal intensity in 31P NMR
spec-tra, but the intensity of those changes depends on the
Fig. 3. Normalized changes of the PPc/Pi ratio in control (K4 spectrum) and N2-treated mycelia (N1 spectrum) in different growth phases.
(p=0.002) compared to N1/k4 values, while at 24, 36, and 44 h, prolonged anoxia has no statistically significant effect.
phase of growth yeast (den Hollander et al., 1981; Campbell-Burk et al., 1987; Beauvoit et al., 1991). In
N. crassa, anoxia does not induce changes in intensi-ties of these signals (Pilatus and Techel, 1991).
The herein obtained results indicate that anoxia leads to considerable changes in the 31P NMR
spec-tra of P. blakesleeanus, but the intensity of these changes varies as a function of both signal origin and development phase. The maximum decrease of intensity, recorded immediately after transition to anaerobic conditions, was observed for the PPc signal. This was expected, considering that in vari-ous stress conditions like changes in medium pH (Greenfield et al., 1987) or osmolarity (Yang et al., 1993), one of the main homeostatic mechanisms is intensive polyphosphate hydrolysis. Decrease in the PPc signal is greater than decrease in the PPt signal, so it can be assumed that in this phase of PPn hydrolysis, apart from exopolyphosphatase activity, endopolyphosphatases also have important role. endopolyphosphatase hydrolyzes inner phosphoan-hydride bonds, cleaving triphosphates from the PPn chain, thus creating a large number of very short PPn chains (kumble and kornberg, 1996). This enzyme was found only in the vacuole (kumble and kornberg, 1996). Considering the fact that in stress conditions vacuolar PPn is hydrolyzed first (kulaev et al., 1999), it can be assumed that this initial and most intensive PPn hydrolysis generally takes place in the vacuole. However, with prolonged anoxia, the PPt signal decreases significantly (Fig. 2B). It is highly unlikely that short chains connect up into longer ones, so this phenomenon could be explained by cessation of endopolyphosphatase activity due to the lack of PPn with a sufficiently long chain. exopolyphosphatases continue to hydrolyze remain-ing chains by cleavremain-ing marginal phosphates, thus shortening PPn without increasing their number. This intensive PPn hydrolysis is not accompanied by increase of Pi signal intensity (Fig 2B). Since the intensities of all other signals in the 31P NMR
spectra also decline, it can be assumed that even in anoxic conditions, part of the Pi obtained by PPn hydrolysis is used for synthesis. Synthesis of ATP may be one of the ways to use this Pi, at least imme-diately after transition to anoxic conditions. To be
specific, the first spectra taken in anaerobic condi-tions show larger decline of the α-ATP relative to the β-ATP signal, pointing to an increase of the ATP/ ADP ratio. This could mean that the cell is trying to sustain ATP concentration at the expense of ADP, providing the means for maintaining basic cellular processes. Since P. blakesleeanus, unlike most fungi, is exclusively an aerobic organism (Galland and Russo, 1979; Wood-Baker, 1955), it is possible that the energy for ATP synthesis is obtained by means of polyphosphate kinase or AMP-phosphotransferase, which can catalyze ATP formation at the expense of PPn (kornberg, 1995; Bonting et al., 1991). This assumption is supported by the fact that polyphos-phate kinase activity was registered in the vacuolar membrane, where most of the PPn hydrolysis takes place in this first stage. Moreover, in prolonged anaerobic conditions, when the intensity of hydroly-sis decreases, the difference in decline of intensity of the α- and β-ATP signals is lost.
Regarding growth phases, 36-h fungi are least sensitive to anoxia, while 32-h and 44-h fungi are most sensitive (Fig 2A). It has been demonstrated that 32-h and 44-h fungi use PPn as an additional source of energy (Živić et al., 2007). It is then under-standable that these growth phases are most affected by anoxia, given that they are most energy-demand-ing. The lack of changes in the spectra of 36-h specimens is much harder to explain. In this spec-trum, only two signals change significantly: Pi signal intensity increases, while UDPG signal intensity decreases. However, these two signals show similar changes in control conditions, except that in the control, the change of Pi signal intensity is smaller, while that of UDPG signal intensity is greater. This means that part or all of the change in these signals is not necessarily induced by anoxia, but by some other, still unknown mechanism.
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ИспИтИвћње ефекћтћ ћноксИје нћ 31Р nMR спектћР гљИве
PHYCOMYCES BLAKESLEEANUS током РћзвИћћ
Марина Станић1, М. Живић2 и Јоана ЗакшевСка2
1Биолошки факултет, Универзитет у Београду, 11000 Београд, Србија 2Институт за општу и физичку хемију, 11000 Београд, Србија
За испитивање ефеката аноксије на мицели јум гљиве Phycomyces blakesleeanus коришћен
је метод 31Р NMR спектроскопије. Највеће про мене су забележене код PPc, NADH и αATP сигнала. Пад интензитета сигнала РРc се може приписати смањењу дужине ланaца и смање њу броја РРc молекула. Мањи пад интензитета
